Challenges of Protein Aggregation during Purification

Careful selection of downstream processing conditions is a must.
Sep 01, 2014
Volume 27, Issue 9

Removal of protein aggregates from biologic APIs is crucial due to their potential to increase  immunogenicity. While most aggregates are formed during upstream operations, and the risk for aggregate formation at the cellular level has increased as titers have increased, if unsuitable conditions are selected for downstream operations, aggregation can also occur during downstream processing.

Aggregation mechanisms
There are essentially two mechanisms leading to aggregation, according to Günter Jagschies, senior director of strategic customer relations for the BioProcess business of GE Healthcare Life Sciences. In one case, the unfolding of a protein from its native to a denatured state may lead to exposure of previously interior hydrophobic structures that can bind to similarly hydrophobic surfaces on other molecules and form aggregates. Alternatively, native proteins may form aggregates via interactions of hydrophobic areas on their outer surface areas. In addition, protein aggregation can refer to a broad range of molecular behaviors, from dimer formation to visible particulates due to noncovalent interactions mediated by hydrophobicity and covalent interactions through pairing of interchain unpaired thiol groups, according to John M. Liddell, head of process sciences with Fujifilm Diosynth Biotechnologies.

“Aggregate formation is generally dependent on the solution conditions (conductivity and pH), protein concentration, and temperature. In addition, aggregates can be soluble when relatively small and insoluble when large, and aggregation may be reversible (from small aggregates) or irreversible (especially when based on denatured protein),” Jagschies notes. The population of incompletely folded and unfolded product and the presence of unpaired cysteine residues can also influence aggregate formation, according to Liddell. Importantly, the conditions that can lead to aggregation can occur during purification processes and need to be avoided or mitigated. “In particular, resolubilization of insoluble aggregates typically requires extra process steps and can both be very costly and compromising on the overall process yield,” adds Jagschies.

Changing the surface chemistry
Downstream processing for recovery and purification is achieved in several steps by exploiting the differences in the affinities, charges, sizes, hydrophobicities, and other physiochemical properties of the therapeutic molecule and impurities, including aggregates. Different combinations of unit operations may be used to recover and purify a given product, including centrifugation, various types of chromatography (e.g., affinity, ion exchange, hydrophobic, etc.), filtration, refolding, and precipitation, according to Ganesh Vedantham, director of process development at Amgen. “All unit operations including chromatography, viral inactivation, viral filtration, and other filtration modes can create conditions that lead to protein aggregation, including shifts in pH and conductivity, high-protein concentrations, and shear,” says Christine Gebski, global purification applications lead at ThermoFisher Scientific.

Strategies used to minimize aggregate formation during downstream processing mainly involve selecting solution conditions and stationary phases (for chromatography) that prevent aggregation and stabilize proteins under the conditions required for purification, sometimes via the addition of excipients or other additives. Recent examples of the latter include the addition of antioxidants such ascorbic acid and glutathione to prevent the generation of sites for aggregation via oxidation of the protein by reactive oxygen, such as aldehyde formation on lysine groups that can then react to form covalent Schiff-based aggregates, according to Shawn Barrett, a scientist with ThermoFisher Scientific. Barrett adds that it has been recently found by some biopharmaceutical manufacturers that the addition of copper at the cell-culture stage leads to a significant reduction in aggregation downstream, although the mechanism is not fully understood at this point.


Analyzing Proteins Using SEC, MALS, and UHPLC

Improving Protein Expression with Novel Systems

More Feature Articles

Centrifugation and filtration
Centrifugation may expose the protein to intense shear forces at the feed-zone location where the feed enters the centrifuge rotor. “Shear can cause protein aggregation directly or indirectly via cell lysis, which leads to aggregation catalyzed by enzymatic reactions,” explains Vedantham. He adds that exposure of proteins at the air-liquid interface during centrifugation and filtration can also result in an increase in soluble and/or insoluble aggregates. Advances in technology, however, are helping. Hermetic sealing of centrifuges now minimizes the air-liquid interface, and appropriate pumps with the flow rates required to minimizing cavitation are available, according to Vedantham. Suitable solution/processing conditions also have been identified that minimize shear stress.

Chromatography is generally the backbone of most purification processes. It also presents opportunities for aggregation to occur. Protein A chromatography is often used as a capture step and provides for the significant removal of impurities from the cell culture. The product is eluted at low pH and then often subjected to a low-pH step for inactivation of potential adventitious viruses. “In both of these unit operations, the protein is exposed to a low-pH environment; some may not be stable under such conditions, leading to aggregation,” Vedantham says.

Ion-exchange chromatography, on the other hand, exploits differences in charges and dipole moments to separate products from impurities. In particular, protein aggregates are often separated from the purified product using a bind-and-elute or flow-through mode of operation. If not controlled, however, Vedantham notes that these operations may also lead to an increase in aggregates, typically due to protein unfolding while the protein is adsorbed on the stationary phase.

Hydrophobic interaction chromatography is generally employed as a polishing step for removing impurities based on hydrophobicity. At the same time, strong hydrophobic interactions between the resin and the product may lead to aggregate formation, according to Vedantham, again due to surface-induced structural perturbation of the product.

“To avoid the undesired interactions that can lead to aggregation during chromatographic purification, it is important to select the appropriate stationary phase with ligands and backbone structures that minimize secondary interactions and a suitable temperature and pH for elution and inactivation,” Vedantham observes. “If aggregation happens spontaneously during the process,” adds Jagschies, “it is not so much the attribute of one of the steps used for purification, but of the protein that is being purified (i.e., its sensitivity to the conditions required). Choosing other conditions or a different method for purification usually helps to avoid the problem.”

Advances in affinity technology are also helping to address some aggregation issues. For example, certain CaptureSelect affinity ligands/resins from Thermo Fisher Scientific have been designed to favor elution under less acidic, more neutral pH conditions, according to Gebski.

Refolding and in-process hold operations
Escherichia coli-based fermentation processes may result in the formation of product trapped as inclusion bodies within the bacterial cell wall.  In such cases, refolding is employed to convert the recovered inclusion bodies to the active product, according to Vedantham. Refolding may lead to formation of aggregated species due to off-pathway reactions.

Finally, during downstream processing of proteins, due to the discrete nature of unit operations execution, product may need to be held after finishing the previous step. Such an in-process hold operation is maintained until the next unit operation is ready to be executed. In some cases, the product may be in an environment that can cause structural perturbation, leading to the formation of aggregates or other undesired product-related species, according to Vedantham. He does note, though, that new methodologies are available for continuous processing that may reduce the time that proteins must spend in such an unfavorable environment.

Benefits of PAT
Advances in process analytical technology (PAT) provide an opportunity for more effective control of protein aggregation in bioprocessing, according to Vedantham. “Dynamic light scattering, which is a non-perturbing technology, has, for example, been used to monitor inclusion body solubilization, protein refolding, and aggregation near the production line of a recombinant protein-based vaccine candidate (1). Use of such real-time process analytical techniques for the monitoring and control of product aggregation will need to be justified, however, with the completion of appropriate cost/benefit analyses,” he says.

Focus on avoidance
Recently, there has been significant effort to prevent aggregation by carefully selecting and/or designing active biologics that are more resistant to aggregation and/or cell lines that lead to the production of proteins with a reduced propensity for aggregation during processing. As part of clone selection early on in development, many companies are looking at the characteristics of the molecule produced by clones with good productivity to understand what purification-relevant features the clone has, according to Jagschies. Clones producing a high aggregate level or that are unstable and have a tendency to self-associate can be deselected at that stage. “Such an approach is much more powerful and efficient than tackling the problem during downstream operations,” Jagschies adds.

GE Healthcare recently announced a licensing agreement with Promosome, a company with a technology for improved protein expression in mammalian cells. “We have hope that Promosome’s technology will be a better approach for controlling expression and minimizing aggregate formation and other issues with product-related impurities. It is very early days, but we are looking forward to, in the foreseeable future, finding good solutions to cell line and expression system construction that will also help the purification process,” states Jagschies.

Currently, high throughput screening techniques are used during the candidate selection phase to select a construct (protein) that is less prone to aggregation, as well as a cell line and cell culture conditions that can further help reduce aggregations, according to Vedantham. Bioinformatics are also being used to tackle the challenge of reducing aggregation. “Application of advanced bioinformatics to molecular design in order to avoid aggregation is increasingly being used. The goal is to improve the surface design of active biologics in order to reduce hydrophobicity and increase hydrophilicity by increasing the number of charged amino acids and eliminating unpaired cysteines,” explains Tibor Nagy, a bioinformatics subject matter expert with Fujifilm Diosynth Biotechnologies. Liddell adds that combining bioinformatics at the design stage with more extensive preformulation screens using high throughput approaches and additional characterization of aggregation propensity using techniques such as light scattering to measure molecular properties (e.g., second virial coefficients) can be effective.

The use of high-pressure to disaggregate and refold proteins is also being explored to control or even reverse aggregation. The technology was developed at the University of Colorado and is currently marketed by BaroFold under the name Pressure Enabled Protein Manufacturing (PreEMT) technology. In January 2013 and November 2012, the company announced agreements with Lonza and Boehringer Ingelheim, respectively, for the evaluation of PreEMT. “It is too early to say what the potential benefits or challenges may be with this technology. In general, however, the avoidance of aggregation seems to be the best route whenever possible,” Jagschies says. He believes that E. coli-based processes may be a target for such methods, because the aggregation that occurs in inclusion bodies has proven difficult and costly to manage.

1. Z. Yu et al., J. Pharm. Sci. 102(12), 4284-4290 (2013).

About the author
Cynthia A. Challener is a contributing editor to BioPharm International.

lorem ipsum